The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.

Elongation factor P (EFP) is a protein that stimulates the peptidyltransferase activity of fully assembled 70 S prokaryotic ribosomes and enhances the synthesis of certain dipeptides initiated by N-formylmethionine. This reaction appears conserved throughout species and is promoted in eukaryotic cells by a homologous protein, eIF5A.Here we ask whether the Escherichia coli gene encoding EFP is essential for cell viability. A kanamycin resistance (Kan R ) gene was inserted near the N-terminal end of the efp gene and was cloned into a plasmid, pMAK705, that has a temperature-sensitive origin of replication. After transformation into a recA ؉ E. coli strain, temperature-sensitive mutants were isolated, and their chromosomal DNA was sequenced. Mutants containing the efp-Kan R gene in the chromosome grew at 33°C only in the presence of the wild-type copy of the efp gene in the pMAK705 plasmid and were unable to grow at 44°C. Incorporation of various isotopes in vivo suggests that translation is impaired in the efp mutant at 44°C. At 44°C, mutant cells are severely defective in peptide-bond formation. We conclude that the efp gene is essential for cell viability and is required for protein synthesis.The most important catalytic function of the ribosome is the synthesis of peptide bonds. A variety of approaches have been used to deduce the components that comprise this catalytic center. The results of in vitro reconstitution studies, photochemical cross-linking of substrates, and mutagenesis of conditionally lethal or antibiotic-resistant phenotypes have implicated domain V of the 23 S rRNA as well as proteins L2, L3, and L4 as the minimum components of this active center (1-6).A surprising finding is that the in vitro reconstituted peptidyltransferase cannot condense all aminoacyl-tRNA template combinations (7). This anomaly is reflected in the fact there is a subsite on domain V of 23 S rRNA that is specific for hydrophobic amino acids (2). In retrospect, it has been known for more than two decades that puromycin, which is one of the most common substrate analogues used to study this reaction, has a special three-dimensional structure (a U shape) that favors peptide-bond synthesis (8). Substitution of the aromatic residue of puromycin by that of other amino acids distorts this structure and drastically impairs peptide-bond synthesis (9). This specificity is reflected in the 50 S catalyzed "fragment" reaction that has been used to deduce the components of the peptidyltransferase catalytic center.Reconstitution studies as well as photoaffinity labeling experiments indicate that several proteins of the 50 S particle enhance peptide-bond synthesis. The assembled peptidyltransferase in the 70 S ribosome catalyzes peptide bonds at a higher rate than does the peptidyltransferase of the 50 S subunit, but does not efficiently condense nonaromatic amino acids (7, 10). In addressing this issue, we asked whether proteins that stimulate reconstitution of translation from homogeneous translation factors enhance the condensation of ...

SummaryShwachman-Diamond Syndrome (SDS) is a multi-system genetic disorder with bone marrow failure. SBDS, the gene associated with SDS, has been postulated to play a role in ribosome biogenesis and RNA processing, but its functions are still unknown. To study whether these pathways are interrupted when Sbds protein is lost, we studied the expression of related genes in patient SBDS)/) cells by an oligonucleotide microarray. We first analysed ribosomal protein (RP) genes, which are normally co-regulated. In SDS, 27 of the 85 RP genes were downregulated. Among the downregulated RP genes, seven are known to be associated with the inhibition of apoptosis. RPS27L, which mediates p53-dependent induction of apoptosis, was the only upregulated RP gene. Interestingly, several genes involved in RP mRNA transcription were downregulated without affecting the expression of genes involved in mRNA degradation, suggesting that the downregulation of the RP gene expression might be at the transcriptional level. Importantly we also found dysregulation of multiple genes involved in rRNA transcription and pre-rRNA processing. We conclude that SDS marrow cells exhibit major dysregulation of RP, RNA processing and RNA transcription genes.

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The syndrome of ataxia-pancytopenia is an autosomal dominant disorder characterized by cerebellar ataxia, peripheral neuropathies, pancytopenia and a predilection to myelodysplastic syndrome and acute myeloid leukemia. The genetic basis of this condition is unknown. We describe a child who presented with ataxia and pancytopenia and was found to have a heterozygous mutation, c.845G>A (Arg282His) in TINF2, a gene recently reported to be mutated in a subset of patients with autosomal dominant dyskeratosis congenita. We propose that some cases of ataxia-pancytopenia may be affected by DC.